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Type VI secretion systems (T6SSs) act as a molecular weapon in interbacterial competition and play an important role in cell-cell interactions. Different species of bacteria use their T6SSs to perform a variety of functions according to ecological niche. Therefore, it is necessary to better understand the T6SS potential ofKlebsiella pneumoniae(K. pneumoniae), a common clinical opportunistic pathogen. Here, we conducted a genomic analysis on the evolution, T6SS, virulence and antimicrobial resistance of 65K. pneumoniaein patients with different infections. And we combined transcriptome analysis after knockout of key gene in T6SS of this species. Results showed that genes encoding a T6SS were present in allK. pneumoniaein this study, and there was no correlation was found between T6SS cluster and carbapenem resistance and virulence genes. Differentially expressed genes (DEGs) including 1298 co-upregulated and 1714 co-downregulated were identified afterhcporvgrGdeletion. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways analysis have demonstrated common changes in quorum sensing, propionate metabolism and other pathways. And we found that the deletion ofhcporvgrGgenes up-regulated of beta-lactam (blaKPC-2) and other resistance genes. Interbacterial competition experiments showed thathcpandvgrGare essential genes for competitive ability of ST11K. pneumoniae. Taken together, the entire study provides further insight into the investigation of T6SS inK. pneumoniaethrough genomic and transcriptomic analysis.ImportanceGram-negative bacteria use T6SS to deliver toxin effectors to interact with neighboring cells for niche advantage.K. pneumoniaeis an opportunistic nosocomial pathogen that often carriers multiple cope T6SSs, but the function of its T6SS has not yet elucidated. Here, we performed a genomic analysis of 65 clinicalK. pneumoniaestrains, in order to explore the relationship between T6SS and virulence and resistance genes. We also study the repertoire after knockout of key gene in T6SS of this species by transcriptomics. It was suggested that T6SS is associated with drug resistance, and its key geneshcpandvgrGare critical for the interspecies competition ofK. pneumoniae.

Peng He,1 Xin Li,2 Xiaohan Guo,1 Xingchen Bian,1 Rui Wang,1 Yue Wang,1 Sijing Huang,1 Mengya Qi,1 Yuanxia Liu,3 Meiqing Feng1 1Department of Biological Medicines & Shanghai Engineering Research Center of Immunotherapeutics, Fudan University School of Pharmacy, Shanghai, 201203, People’s Republic of China; 2Institute of Antibiotics, Huashan Hospital, Fudan University, Shanghai, People’s Republic of China; 3Shanghai Municipal Hospital of Traditional Chinese Medicine, Shanghai University of Traditional Chinese Medicine, Department of Pathology, Shanghai, People’s Republic of ChinaCorrespondence: Meiqing Feng, Department of Biological Medicines & Shanghai Engineering Research Center of Immunotherapeutics, Fudan University School of Pharmacy, Shanghai, 201203, People’s Republic of China, Tel +86 21 51980035, Email fmq@fudan.edu.cn Yuanxia Liu, Shanghai Municipal Hospital of Traditional Chinese Medicine, Shanghai University of Traditional Chinese Medicine, Department of Pathology, Shanghai, People’s Republic of China, Email lyuyx@126.comIntroduction: LYSC98 is a novel vancomycin derivative used for gram-positive bacterial infections. Here we compared the antibacterial activity of LYSC98 with vancomycin and linezolid in vitro and in vivo. Besides, we also reported the pharmacokinetic/pharmacodynamic (PK/PD) index and efficacy-target values of LYSC98.Methods: The MIC values of LYSC98 were identified through broth microdilution method. A mice sepsis model was established to investigate the protective effect of LYSC98 in vivo. Single-dose pharmacokinetics of LYSC98 was studied in thigh-infected mice and liquid chromatography–tandem mass spectrometry (LC-MS/MS) method was used to determine LYSC98 concentration in plasma. Dose fractionation studies were performed to evaluate different PK/PD indices. Two methicillin-resistant Staphylococcus aureus (MRSA) clinical strains were used in the dose ranging studies to determine the efficacy-target values.Results: LYSC98 showed a universal antibacterial effect in Staphylococcus aureus with a MIC range of 2– 4 μg/mL. In vivo, LYSC98 demonstrated distinctive mortality protection in mice sepsis model with an ED50 value of 0.41– 1.86 mg/kg. The pharmacokinetics results displayed maximum plasma concentration (Cmax) 11,466.67− 48,866.67 ng/mL, area under the concentration–time curve from 0 to 24 h (AUC0– 24) 14,788.42− 91,885.93 ng/mL·h, and elimination half-life (T1/2) 1.70– 2.64 h, respectively. Cmax/MIC (R2 0.8941) was proved to be the most suitable PK/PD index for LYSC98 to predict its antibacterial efficacy. The magnitude of LYSC98 Cmax/MIC associated with net stasis, 1, 2, 3 and 4 - log 10 kill were 5.78, 8.17, 11.14, 15.85 and 30.58, respectively.Conclusion: Our study demonstrates that LYSC98 is more effective than vancomycin either in killing vancomycin-resistant Staphylococcus aureus (VRSA) in vitro or treating S. aureus infections in vivo, making it a novel and promising antibiotic. The PK/PD analysis will also contribute to the LYSC98 Phase I dose design.Keywords: LYSC98, vancomycin derivative, pharmacokinetics and pharmacodynamics, Staphylococcus aureus, murine thigh infection model

Pharmacometrics is an emerging science that interprets drug, disease, and trial information in a mathematical fashion to inform and facilitate efficient drug development and/or regulatory decisions. Pharmacometrics study is increasingly adopted in the regulatory review of new antimicrobial agents. We summarized the 31 antimicrobial agents approved by the US Food and Drug Administration (FDA) and the 26 antimicrobial agents approved by European Medicines Agency (EMA) from January 2001 to May 2019. We also reviewed recent examples of utilizing pharmacometrics to support antimicrobial agent’s registration in China, including modeling and simulation methods, effects of internal/external factors on pharmacokinetic (PK) parameters, safety and efficacy evaluation in terms of exposure‐response analysis, refinement of the wording of product labeling and package leaflet, and possible postmarketing clinical trial. Ongoing communication among regulator, academia, and industry regarding pharmacometrics is encouraged to streamline and facilitate the development of new antimicrobial agents. The industry can maximize its benefit in drug development through continued pharmacometrics education/training.

Levornidazole is a novel nitroimidazole antimicrobial agent active against anaerobes. We aimed to investigate the pharmacokinetic (PK) profile of levornidazole after single and multiple oral doses of levornidazole tablets in healthy Chinese subjects and propose the dosing regimen based on pharmacokinetic/pharmacodynamic (PK/PD) analysis. A single-center, randomized, double-blind, placebo-controlled study was conducted with a single ascending dose (250, 500, 1000, and 1500 mg) and multiple doses of 500 mg levornidazole q12h for 7 days. Food effect on PK and absolute bioavailability were investigated at the 500 mg dose level. Blood and urine samples were collected to determine the PK parameters of levornidazole. The probability of target attainment (PTA) and cumulative fraction of response (CFR) were calculated by Monte Carlo simulation to predict the clinical efficacy of levornidazole tablets. Plasma concentration reached peak about 0.5 h after single dose (250–1500 mg) of levornidazole tablets. The maximal concentration (Cmax) and exposure (AUC0–∞) of levornidazole increased linearly with dose. High-fat diet did not affect the absorption extent of levornidazole tablets. The absolute oral bioavailability of levornidazole tablets was 98.3% ± 7.6%, associated with large apparent volume of distribution (48.68 ± 4.92 l) and long half-life (11.93 ± 1.28 h). The urinary excretion of levornidazole was 7.99%. Levornidazole, administered at either 500 mg q12h or 750 mg q24h, achieved a CFR > 95.4% and PTA > 99% for B. fragilis (minimum inhibitory concentration ≤ 1.0 mg/l) infections. Levornidazole tablets are absorbed rapidly and completely and distributed extensively with a long half-life and low urinary excretion after a single dose or multiple doses in healthy Chinese subjects. Levornidazole tablets can be taken with or without food. Levornidazole tablets 500 mg q12h and 750 mg q24h are expected to achieve the desired efficacy in B. fragilis infections. Trial registration number CTR20160786 at http://www.chinadrugtrials.org.cn/ .

Objectives Polymyxin B is a last-line antibiotic for multidrug-resistant gram-negative bacterial infections. However, limited safety and pharmacokinetic information is available. We investigated the safety and pharmacokinetics of intravenous polymyxin B in healthy subjects. Methods An open-label, single-dose clinical trial was conducted in healthy Chinese subjects. Polymyxin B (sulphate) was administered intravenously at 0.75 or 1.5 mg/kg (n = 10 per dose, 5 males and 5 females) to examine the safety and pharmacokinetics. Results One female subject in the 1.5-mg/kg group discontinued due to abdominal pain during administration. The most frequently reported adverse events were perioral paraesthesia, dizziness, and numbness of extremities (7/10 subjects in the 0.75-mg/kg group, all subjects in the 1.5-mg/kg group). All neurotoxicity-related events dissipated without treatment within a maximum of 23 h. Notably, abdominal pain (3/5) and vulvar pruritus (2/5), colpitis (2/5) or abnormal uterine bleeding (1/5) were reported in female subjects receiving the 1.5-mg/kg dose. In the 0.75-mg/kg group, the total clearance, volume of distribution and half-life of polymyxin B were 0.028±0.002 L/h/kg, 0.219±0.023 L/kg and 5.44±0.741 h, respectively; similar values were observed in the 1.5-mg/kg group. Urinary recovery was 3.7 ± 1.1% and 8.1 ± 1.3% in the 0.75- and 1.5-mg/kg groups, respectively. Population pharmacokinetics of polymyxin B was consistent with a three-compartment model. The clearance and distribution of the central compartment were 0.027 L/h/kg and 0.071 L/kg, respectively. Conclusions This study is the first to examine the safety and pharmacokinetics of polymyxin B in healthy subjects. Our results highlight that acute toxicity is a dose-limiting factor for intravenous polymyxin B.

Supported Pd-Cu NO3− reduction (NO3R) under H2 atmosphere is considered as a promising way to achieve NO3− removal directly in water through converting into N2, which is a harmless product in environment. However, desirable NO3R kinetics and N2 selectivity remain challenges. In this research, 2.5 wt% Cu and 10 wt% Pd are loaded on various oxides supports and combine with electrolytic in-situ H2 generation for NO3R. Pd10%Cu2.5%/TiO2 demonstrates a faster NO3R rate constant than Pd10%Cu2.5%/γ-Al2O3 and even greater than Pd10%Cu2.5%/CeO2. Pd10%Cu2.5%/γ-Al2O3 obtains better N2 selectivity than the other two. Crucially, the oxygen vacancies (VO) existing in TiO2 and CeO2 can reduce the nearby Cu2+ to Cu+, also Cu2+ can attract electrons from Pd0 to form Cu+ on the γ-Al2O3 surface. Verified by experiments and density functional (DFT) calculations, these Cu+ based active sites on the surface of substrates are vital for effective NO3− adsorption and high N2 selectivity.

The mass transfer on the catalyst surface has a great influence on the selectivity of electrocatalytic nitrate reduction to nitrogen. In this study, a Pd-Cu adsorption confined nickel foam cathode is designed in the absence of both proton exchange membranes and chloride ions. The repulsion of the cathode enables intermediate products such as nitrite to accumulate in the confined region, resulting in an increase in the possibility of a second-order reaction to form nitrogen. The system can obtain more than 92% continuous N

Backgrounds: Polymyxin B， which is a last line antibiotic for multi-drug resistant gram-negative bacteria, has been reported to induce skin hyperpigmentation in patients during treatments. 8~15% or more cases were reported hyperpigmentation, especially in head and neck, seriously influencing the compliance of patients. To investigate the mechanism of hyperpigmentation is beneficial for intervention strategy and improve compliance. Methods: The melanoma cells, SK-MEL-2 cells were employed in the present study to verify whether polymyxin B treatment could directly induce hyperpigmentation. Melanin contents were measured by hot alkali lysis method and tyrosinase were quantified by dopaquinone oxidation method. The real-time quantitative PCR was applied to measure the mRNA levels of melanogenesis-related genes to investigate the possible signaling pathway involved in the polymyxin B induced hyperpigmentation. Results: The melanin content and tyrosinase activity were up-regulated after 5 μg/mL polymyxin B treatment in SK-MEL-2 cells at 48 hr. and 72 hr. The mRNA levels of melanogenesis-related genes were up-regulated, including PI3K, Akt, GSK-3β, CREB, MITF in the polymyxin B treatment group compared with control group at 48 hr. (p

Polymyxin-based combination therapy is commonly used to treat carbapenem-resistant

LYSC98 is a vancomycin derivative used for gram-positive bacterial infections therapy. We reported the pharmacokinetic/pharmacodynamic (PK/PD) targets of LYSC98 against Staphylococcus aureus using a murine thigh infection model. Three Staphylococcus aureus strains were utilized. Single-dose plasma pharmacokinetics of LYSC98 were determined in infected mice after the tail vein injection of 2, 4, and 8mg/kg. The results showed maximum plasma concentration (Cmax) 11466.67 - 48866.67 ng/mL, area under the concentration-time curve from 0 to 24 h(AUC0-24) 14788.42 -91885.93 ng/mL·h, and elimination half-life(T1/2) 1.70-2.64 h, respectively. The Cmax (R2 0.9994) and AUC0-24 (R2 0.981) were positively correlated with the dose of LYSC98 in the range of 2-8 mg/kg.Dose fractionation studies using total doses of 2 to 8 mg/kg administered with q6h, q8h, q12h, and q24h were performed to evaluate the correlation of different PK/PD indices with efficacy. Sigmoid model analysis showed Cmax/MIC (R2 0.8941) was the best PK/PD index to predict the efficacy of LYSC98. In the dose ranging studies, two Methicillin-resistant Staphylococcus aureus (MRSA) clinical strains were used to infect the mice and 2-fold-increasing doses (1 to 16 mg/kg) of LYSC98 were administered. The magnitude of LYSC98 Cmax/MIC associated with net stasis, 1, 2, 3 and 4 - log10 kill were 5.78, 8.17, 11.14, 15.85 and 30.58, respectively. The results of this study showed LYSC98 a promising antibiotic with in vivo potency against MRSA, and will help in the dose design of phase one study for LYSC98.

Antimicrobial resistance is a major global health challenge. As few new efficacious antibiotics will become available in the near future, peptide antibiotics continue to be major therapeutic options for treating infections caused by multidrug-resistant pathogens. Rational use of antibiotics requires optimisation of the pharmacokinetics and pharmacodynamics for the treatment of different types of infections. Toxicodynamics must also be considered to improve the safety of antibiotic use and, where appropriate, to guide therapeutic drug monitoring. This review focuses on the pharmacokinetics/pharmacodynamics/toxicodynamics of peptide antibiotics against multidrug-resistant Gram-negative and Gram-positive pathogens. Optimising antibiotic exposure at the infection site is essential for improving their efficacy and minimising emergence of resistance.

BackgroundAcinetobacter baumanniiis a common nosocomial pathogen that poses a huge threat to global health. Owing to the severity ofA. baumanniiinfections, it became necessary to investigate the epidemiological characteristics ofA. baumanniiin Chinese hospitals and find the reasons for the high antibiotic resistance rate and mortality. This study aimed to investigate the epidemiologic and genetic characteristics ofA. baumanniiisolated from patients with hospital acquired pneumonia (HAP), bloodstream infection (BSI) and urinary tract infection (UTI) in China and uncover potential mechanisms for multi-drug resistance and virulence characteristics ofA. baumanniiisolates.ResultsAll isolates were classified into two primary clades in core gene-based phylogenetic relationship. Clonal complex 208 (CC208) mainly consisted of ST195 (32 %) and ST208 (24.6 %). CC208 and non-CC208 isolates had carbapenem resistance rates of 96.2 and 9.1 %, respectively. Core genes were enriched in ‘Amino acid transport and metabolism’, ‘Translation’, ‘Energy production and conversion’, ‘Transcription’, ‘Inorganic ion transport and metabolism’ and ‘Cell wall/membrane/envelope synthesis’. Most isolates possessed virulence factors related to polysaccharide biosynthesis, capsular polysaccharide synthesis and motility. Eleven isolates belong to ST369 or ST191 (oxford scheme) all had the virulence factorcap8Eand it had a higher positive rate in UTI (35.3 %) than in BSI (18.9 %) and HAP (12.9 %). ABGRI1 antibiotic resistance islands were responsible for streptomycin, tetracycline and sulfonate resistance. TheblaOXA−23gene was the most probable cause for carbapenem resistance, although theblaOXA−66gene with nonsynonymous SNPs (F82L, I129L) was not.ConclusionsA. baumanniiis a genomically variable pathogen that has the potential to cause a range of infectious diseases. There is high proportion of carbapenem resistance in isolates from all three infection sites (HAP, BSI and UTI), which can be attributed to theblaOXA−23gene. CC208 is the predominant clone inblaOXA−23-carryingA. baumanniithat should be monitored. Virulence factors involving bacteria motility and polysaccharide biosynthesis which are widespread in clinicalA. baumanniistrains deserve our attention.

Aims: Polymyxin-based combination therapy is often used to treat carbapenem-resistant Acinetobacter baumannii (A. baumannii) infections. Although sulbactam is intrinsically active against A. baumannii, few studies have investigated colistin/sulbactam combinations against carbapenem-resistant A. baumannii. Methods: Whole genome sequencing was undertaken on eight carbapenem-resistant (colistin-susceptible) isolates of A. baumannii from Chinese patients. Bacterial killing of colistin and sulbactam, alone and in combination, was examined with checkerboard (all isolates) and static and dynamic time-kill studies (three isolates). In the dynamic studies, antibiotics were administered in various clinically-relevant dosing regimens that mimicked patient pharmacokinetics. Results: The eight isolates consisted of ST195, ST191 and ST208 belonging to clonal complex 208, which is the most epidemic clonal type of A. baumannii globally. All isolates possessed Acinetobacter-derived cephalosporinase (ADC-61 or ADC-78) and seven of eight isolates contained the carbapenem-resistance gene blaOXA-23. The colistin/sulbactam combination was synergistic against two of eight isolates in checkerboard studies. In time-kill studies, rapid bacterial killing of ca. 3–6 log10 CFU/mL was observed with colistin monotherapy, followed by steady regrowth. Sulbactam monotherapy was generally ineffective. Substantially enhanced bacterial killing was observed with colistin/sulbactam combinations in both static and dynamic models, especially with the higher sulbactam concentration (2 g) and/or longer sulbactam infusion time (2 hours) in the dynamic model. Conclusions: This study was the first to use a pharmacokinetics/pharmacodynamics model to investigate synergistic activity of colistin/sulbactam combinations against A. baumannii. It showed that clinically-relevant dosing regimens of colistin combined with sulbactam may substantially improve bacterial killing of multidrug-resistant and carbapenem-resistant A. baumannii.

Background: A robust and rapid method for therapeutic drug monitoring (TDM) is urgently needed for polymyxin B, which is a last-line antibiotic for multidrug-resistant gram-negative bacteria infection. Methodology: A 3-min run of LC–MS/MS method was established to determine the main components of polymyxin B (polymyxin B1 and B2) in human plasma or urine. Solid-phase extraction was employed to eliminate the matrix effect from complicated samples from patients. Results: The calibration range was 0.050–5.00 and 0.0110–0.549 μg/ml for polymyxin B1 and B2, respectively, in plasma and urine. The precision and accuracy of quality controls, matrix effect, extraction recovery and stability were all validated and satisfied with the ICH requirements. The method was successfully applied to a pharmacokinetic study in healthy subjects and TDM in patients. Conclusion: The rapid LC–MS/MS method was validated for polymyxin B in plasma and urine, and robust for TDM.

Colistin-based combination therapy has become an important strategy to combat the carbapenem-resistant Acinetobacter baumannii (CRAB). However, the optimal dosage regimen selection for the combination with maximum efficacy is challenging. Checkerboard assay was employed to evaluate the synergy of colistin in combination with meropenem, rifampin, fosfomycin, and minocycline against nine carbapenem-resistant A. baumannii isolates (MIC of meropenem [MICMEM], ≥32 mg/liter) isolated from Chinese hospital-acquired pneumonia (HAP) patients. A static time-kill assay, in vitro dynamic pharmacokinetic/pharmacodynamic (PK/PD) model, and semimechanistic PK/PD modeling were conducted to predict and validate the synergistic effect of the most efficacious combination. Both checkerboard and static time-kill assays demonstrated the superior synergistic effect of the colistin-meropenem combination against all CRAB isolates. In the in vitro PK/PD model, the dosage regimen of 2 g meropenem daily via 3-h infusion combined with steady-state 1 mg/liter colistin effectively suppressed the bacterial growth at 24 h with a 2-log10 decrease, compared with the initial inocula against two CRAB isolates. The semimechanistic PK/PD model predicted that more than 2 mg/liter colistin combined with meropenem (2 g, 3-h infusion) was required to achieve the killing below the limit of detection (

Co3O4 rod and sheet-like films with different crystal facet exposure and Co2+/Co3+ ratio are synthesized on Ti substrate through a facile hydrothermal method with the adjusting of hydrothermal pressure and duration. Low pressure and short hydrothermal duration is likely to obtain rod-like film with almost pure (220) facet exposure, while the sheet-like film with the mixture of (220) and (222) facets is formed with relatively high pressure. Moreover, the Co2+/Co3+ ratio is 2.067 for Co3O4 rods and 1.717 for Co3O4 sheets. The FT-IR and the XPS results indicate that the improvement of Co2+/Co3+ ratio possibly results from the doping of CO32−/OH−, and the introduction of oxygen vacancies. The films are employed as cathodes for electrocatalytic NO3−-N reduction. Because of the larger electrochemical active area and the lower internal resistance, better conversion of NO3−-N to NH4+-N is obtained by Co3O4 sheets film at constant voltage condition. However, the results are opposite when the constant current is applied. The larger exposure of (220) facet and the more amount of Co2+ and oxygen vacancies result in the better NO3−-N removal and NH4+-N conversion kinetics of Co3O4 rods film than that of Co3O4 sheets.

Carbapenem-resistant Acinetobacter baumannii (CRAB) is an important clinical threat. Combination therapy that exerts a synergistic effect has become a potential solution to combat CRAB. However, choosing an optimal combination regimen is challenging. A dynamic in vitro pharmacokinetic/pharmacodynamic (PK/PD) model that can simulate the pharmacokinetic profiles of antibiotics provides a powerful tool to compare antibacterial responses to different clinical dosage regimens. In this study, the synergistic effect of the combination of meropenem and colistin was tested in 12 clinical CRAB isolates from Chinese patients using the chequerboard technique. The antibacterial effect was investigated in an in vitro PK/PD diffusion model by simulating different dosage regimens: meropenem monotherapy (0.5 g with 0.5-h infusion or 1 g with 3-h infusion); colistin monotherapy (fixed unbound concentration maintained at 0.25, 0.5 or 1 mg/L); and combination of meropenem and colistin. The chequerboard method showed that the combination of meropenem and colistin had synergistic effects against all 12 isolates, with fractional inhibitory concentration indices (FICIs) of ≤0.5. Moreover, the dynamic in vitro PK/PD model demonstrated that for clinical CRAB isolates with a meropenem MIC of 128 mg/L, the combination (meropenem 1 g with 3-h infusion combined with colistin maintained at 1 mg/L) could achieve 3.8 log10 killing after 24 h, whereas monotherapy was unable to provide such an antibacterial effect. Taken together, these results suggest that the combination of meropenem and colistin might be a promising therapy against CRAB.

Colistin-based combination therapy has become an important strategy to combat the carbapenem-resistant Acinetobacter baumannii (CRAB). However, the optimal dosage regimen selection for the combination with maximum efficacy is challenging. Checkerboard assay was employed to evaluate the synergy of colistin in combination with meropenem, rifampin, fosfomycin, and minocycline against nine carbapenem-resistant A. baumannii isolates (MIC of meropenem [MIC(MEM)], ≥32 mg/liter) isolated from Chinese hospital-acquired pneumonia (HAP) patients. A static time-kill assay, in vitro dynamic pharmacokinetic/pharmacodynamic (PK/PD) model, and semimechanistic PK/PD modeling were conducted to predict and validate the synergistic effect of the most efficacious combination. Both checkerboard and static time-kill assays demonstrated the superior synergistic effect of the colistin-meropenem combination against all CRAB isolates. In the in vitro PK/PD model, the dosage regimen of 2 g meropenem daily via 3-h infusion combined with steady-state 1 mg/liter colistin effectively suppressed the bacterial growth at 24 h with a 2-log(10) decrease, compared with the initial inocula against two CRAB isolates. The semimechanistic PK/PD model predicted that more than 2 mg/liter colistin combined with meropenem (2 g, 3-h infusion) was required to achieve the killing below the limit of detection (